Project Summary Ion channels in cardiomyocytes determine the shape, duration, and frequency of the heartbeat. Many factors that alter the function of channels cause cardiac disease in humans, including genetic mutations, adverse drug side-effects, or changes to cell-signaling pathways that regulation channel activity. This proposal is based on new insights into the regulation of two ion channel types that are critical to the function of the human heart: (1) The Kir2 channels that pass IK1, a current that contributes to the long-lasting plateau phase of the cardiac action potential and (2), the voltage-gated sodium channel, NaV1.5 that mediates INa, the current that underlying the rapid upstroke of the action potential. The activity of Kir2 channels (Kir2.1 to Kir2.4) is dependent on the ubiquitous, anionic membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 regulates multiple cellular processes and proteins, including TRP, Kir and at least some voltage-gated potassium channels. Significant research effort has characterized the structure-function relationship of PIP2 and Kir2 channels, including a crystal structure of PIP2 in complex with Kir2.2. Many disease-associated mutations and cell signaling pathways alter the function of Kir2 channels by interfering with the interaction between the channel and PIP2. Much less is known about channel- regulation by SUMOylation, a post translational modification (PTM) pathway that culminated in the covalent modification of channels by the an ~100-amino acid SUMO protein. Although SUMOylation regulates numerous ion channel proteins in a range of excitable tissues, including cardiomyocytes, neurons, and pancreatic ?-cells, little is known about the cellular stimuli that impact SUMOylation, or about the molecular basis for the action of SUMO on channel proteins. We recently showed that neuronal NaV1.2 channels are rapidly SUMOylated in response to acute hypoxia, increasing current through the channels. We were intrigued to understand if this mode of regulation applied to NaV1.5 and PIP2-dependent Kir2 channels in cardiomyocytes. Data from our preliminary studies in heterologous systems and human ventricular cardiomyocytes, derived from induced pluripotent stem cells (iPS-CMs), support five novel findings: (1) Kir2.1 and 2.4 channels are SUMO-regulated; (2) the effect of SUMO on these channels is PIP2-dependent; (3) NaV1.5 channels are SUMO-regulated; (4) SUMOylation of cardiac Nav1.5 increases rapidly in response to acute hypoxia; (5), NaV1.5 channel activity is PIP2-dependent. Based on these preliminary observations, we propose the hypothesis that Kir2 and NaV1.5 channels are SUMO-regulated in a PIP2-dependent manner and describe two experimental aims designed to characterize these findings and test our hypothesis. By studying Kir2 and NaV1.5 channels, we expect to understand the role of SUMO in the regulation of Kir2 channels and the role of PIP2 in the regulation of NaV1.5 channels. Further, we expect to demonstrate that SUMO-mediated effects on channels occur via PIP2, providing a unifying molecular mechanism for all SUMO- and PIP2-dependent ion channels.